The aim of this research is to obtain structural information on H-Y antigen, a constitutive cell-surface component of tissues from vertebrates possessing heterogametic gonads. Its biological importance is emphasized by the results of comparative serological and immunological studies which provide evidence that the molecule is highly conserved in vertebrate evolution. In mammals, the H-Y antigen structural gene or a gene regulating its expression is located on the Y chromosome, and is identified as the testis-determining gene. Thus, its biological function resides in its role as the inducer of testicular organogenesis. Cultures of Daudi human male Burkitt lymphoma cells excrete H-Y antigen into the culture medium. This excretion may be related to the loss of expression of membrane-associated Beta2-microglobulin-HLA antigen dimers functioning as the membrane anchorage sites for H-Y antigen. Thus, we have undertaken to isolate and purify amounts of H-Y antigen from Daudi cell cultures sufficient to undertake studies of the carbohydrate and protein structure of pure H-Y antigen. The carbohydrate studies will include methylation analysis and periodate oxidation-Smith degradation. The carbohydrate portions of any glycopeptides obtained will be examined using specific exo- and endoglycosidase cleavage techniques. Protein sequence determinations will be performed on peptides obtained by specific enzymatic and chemical cleavage of H-Y antigen. Purified peptides will be tested for biological activity as inducers of testicular differentiation in organ cultures of indifferent gonads obtained from bovine embryos. The studies proposed here are likely to have direct application in studies of the biological and biochemical mechanisms of action of H-Y antigen, an important prototype of differentiation antigens.